The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
Fluorescence measurements are an invaluable tool for a wide variety of applications in various fields, including analytical chemistry, biochemistry, cell biology, physiology, cardiology, photochemistry, environmental science, and other basic science and clinical research. A primary advantage of fluorescence measurement over absorption measurement is its high selectivity and sensitivity. For example, the dynamics of protein folding and unfolding can be studied using single-molecule fluorescence detection. High throughput fluorescence screening can be performed to find potential drug leads from an extensive library of compounds. Fluorescence emission from ions can be used to quantify their local concentrations in living cells. Membrane structure and function can be studied with fluorescence probes. Drug delivery and its treatment effects can be monitored in living biological systems. Minute traces of fluorescent materials can be detected and identified for forensic science and homeland security. Binding properties of biochemical species can be monitored in real time and in situ by fluorescence measurements.
In an attempt to address the variety of fluorescence-based measurements, some detection instruments have been used in both research institutions and industry. One such instrument employs fluorescence microscopy, which has become one of the most rapidly expanding microscopy techniques employed today, both in medical and biological sciences. In fluorescence microscopy, fluorescent dyes are used to label specific subcellular components, which can then be optically imaged. Similarly, a number of microscopes and fluorescence accessories have been developed, such as laser scanning confocal microscopes and multiphoton fluorescence microscopes, to aid in such imaging. Different from fluorescence imaging, flow cytometers have been used to measure the total fluorescence from each cell to enable large populations of cells to be studied, thereby providing quantitative information on many important biological processes (e.g. receptor expression, analysis of intracellular proteins, targeted drug uptake, etc.). Despite the broad applications of fluorescence measurements and a long history of the development of various kinds of fluorescence detection systems, the basic detection mechanism remains unchanged until now.
One of the most important considerations for a fluorescence detection system is to separate fluorescence signals from excitation light. As a basic fluorescence nature, fluorescence emission occurs at a longer wavelength due to the Stokes shift when certain molecules have absorbed excitation photons of shorter wavelengths. Emission filters are often used to screen out the stray light such as Rayleigh and Raman scatter from the sample under excitation and from other components in the optical path, allowing primarily the wavelength of fluorescence light specific to the sample to pass through. Often limited by the small Stokes shift, some fluorescence signal has to be sacrificed in order to completely block the stray light, thus preventing a whole fluorescence spectrum from being observed. In addition, both absorption and emission are unique characteristics of a particular molecule. Thus, with a single excitation wavelength, such as a laser source, only a limited number of fluorophores that have absorption matched with the excitation wavelength can be excited and thus detected. Even if some broadband light sources, such as xenon lamps, are sometimes used for excitation, excitation filters are often used, which allows a selected band of light energy to pass through and excite the sample while blocking other wavelengths, especially those in the emission spectrum. Therefore, the types of fluorophores that can be simultaneously excited are limited in this case. These drawbacks in conventional fluorescence measurements have not only reduced the detection speed and sensitivity, but have also limited the selection of detectable fluorescent markers.
According to the principles of the present teachings, a fluorescence detection system for testing a sample having at least one fluorophore is provided having advantageous construction. The fluorescence detection system comprises a white light generation system outputting a white light pulse. The white light pulse has a first frequency range and a first time duration. The white light pulse excites the at least one fluorophore of the sample to emit a fluorescence. The fluorescence has a second frequency range and a second time duration, wherein the first time duration is less than the second time duration. A time-resolving detector receives the fluorescence and at least a portion of the white light pulse and separates the fluorescence from the portion of the white light pulse.
Further areas of applicability will become apparent from the description provided herein. It should be understood that the description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.